Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. The virus-encoded bZIP family protein K8 plays an important role in viral DNA replication in both viral reactivation and de novo infection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here, we report that K8 is an RNA binding protein that also associates with many other proteins, including other RNA binding proteins. Many protein-protein interactions involving K8 are mediated by RNA. Using a UV cross-linking and immunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, including both viral (PAN, T1.4, T0.7, etc.) and cellular (MALAT-1, MRP, 7SK, etc.) RNAs. An RNA binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 to bind to many noncoding RNAs, as well as viral DNA replication during de novo infection, suggesting that the K8 functions in viral replication are carried out through RNA association. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication during de novo infection.

IMPORTANCE Genomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs.

卡波济氏肉瘤相关疱疹病毒（KSHV）裂解复制和新鲜细胞持续原发感染对于病毒致瘤性至关重要。病毒编码的bZIP家族蛋白K8在病毒激活和从头感染中在病毒DNA复制中起重要作用。K8在病毒生命周期中发挥功能作用的机制尚不清楚。在这里，我们报道K8是一种RNA结合蛋白，还与许多其他蛋白（包括其他RNA结合蛋白）相关。RNA介导了许多涉及K8的蛋白质相互作用。使用UV Ç罗斯-l着墨和我mmuno p沉淀（CLIP）程序与高通量测序相结合，鉴定了与BCBL-1细胞中K8相关的RNA，包括病毒（PAN，T1.4，T0.7等）和细胞（MALAT-1， MRP，7SK等）RNA。定义了K8中的RNA结合基序，并且该基序的突变消除了K8与许多非编码RNA结合的能力以及从头进行的病毒DNA复制感染，表明病毒复制中的K8功能是通过RNA结合来实现的。详细研究了K8和相关的T1.4 RNA的功能，结果表明T1.4介导K8与ori-Lyt DNA的结合。T1.4-K8复合物与KSHV ori-Lyt DNA物理结合，并募集其他蛋白质和辅因子来组装复制复合物。T1.4的消耗消除了原发感染中的DNA复制。这些发现为从头感染期间K8与T1.4 RNA协同调节KSHV DNA复制中的作用提供了机械方面的见解。

重要性哺乳动物转录组的全基因组分析表明，以前注释为非编码区的大部分序列实际上已转录并产生稳定的RNA。大量非编码RNA的出现表明功能性RNA-蛋白质复合物（例如核糖体或剪接体）不是最后一个核糖体生物的古老遗迹，但可以很好地适应生物学中的调节作用。K8由于其独特的特性而令人困惑，例如在不具有DNA结合能力的基因表达和DNA复制中的多种调节作用。这项研究通过证明K8是与DNA结合并与非编码RNA协同启动DNA复制的RNA结合蛋白，揭示了其调控作用的潜在机制。建议提供许多K8功能（如果不是全部的话），